The insulin-like growth factor (IGF)-I system is critical for all phases of human development, growth, and maturation. While it is well established that IGF-1 production is highly dependent on growth hormone (GH) during postnatal life, mechanisms of regulation are still poorly understood, as reflected by the 1-2% children who drop below the normal growth curves, many of whom respond poorly to GH (including therapeutic GH) and are IGF-1 deficient. GH regulates the production of IGF-1 primarily through activation of the GH receptor (GHR) signaling cascades. The identification of rare mutations of STAT5B (signal transducer and activator of transcription 5B) in children who resembled patients carrying GHR defects, with severe postnatal growth failure, GH insensitivity (GHI) and severe IGF deficiency (IGFD), confirmed the critical importance of STAT5b, over that of the STAT1, 3 and 5a signaling pathways, in IGF-1 production and human growth. Patients with STAT5b deficiency, moreover, also presented with histories of immune dysregulation, indicating the importance of STAT5b in many cytokine systems. These clinical presentations of a STAT5b deficiency state are unique to humans. Of note, the presence of the closely related STAT5a (which shares >90% similarity with STAT5b at the protein level) could not compensate for human loss of STAT5b functions, thereby providing the first compelling in vivo evidence for human STAT5b and STAT5a as having distinct and non-redundant functions despite their high degree of identity. The hypothesis of this proposal, based on clinical observations, is that GH regulation of IGF-1 production in humans is mediated predominantly, if not exclusively, through STAT5b. This proposal will address how STAT5b transduces GH signals to regulate IGF-1 production by investigating molecular mechanisms of STAT5b actions, from structure/function to cell signaling, and their relationship to clinical endocrinology and immunology, using identified mutations as human functional knockouts. The Specific Aims will (1) decipher the specificity of human GHR-STAT5b structural interactions, the critical first step in regulating IGF-1 production; (2) delineate STAT5b properties necessary for IGF-1 production from those necessary for immune functions; (3) determine whether cross talk with other cytokine signaling pathways modulate STAT5b- dependent activation IGF-1 production. Humanized mouse model can then be generated to recapitulate the human STAT5b deficiency condition. Exome sequence analysis of unresolved clinical cases of severe IGFD with immune deficiencies has revealed potential new mechanisms of modulating STAT5b actions. At the conclusion of these studies, this proposal will have identified and confirmed the mechanism(s) for specificity of GHR-STAT5b interactions, identified new mechanism(s) for modulating STAT5b activation and regulation of IGF-1 production, and identified new targets for analyzing GHI and IGFD syndromes. In the long term, these results will provide the basis for improved diagnosis and therapy management of affected children.